JP6040157B2 - Coelenterazine derivatives and methods of use - Google Patents

Description

(Cross-reference of related applications)
This application claims priority to US Patent Application No. 61 / 409,413, filed Nov. 2, 2010, which is hereby incorporated by reference in its entirety.
The present invention provides compounds and methods for assaying for the presence and activity of enzymes.

  The presence and activity of the enzyme can be used to determine the health or metabolic state of the cell. Enzymes can also be markers for specific cell types because the appearance and activity of an enzyme is often unique to a particular cell. For example, the activity of a certain enzyme can be used to distinguish cells from bacteria, plants or animals, or to identify the identity of the tissue from which the enzyme is derived.

  Glycosidases, also known as glycoside hydrolases, catalyze the hydrolysis of glycosidic bonds and produce two smaller sugars. Glycosidases include natural degradation, including biomass degradation such as cellulose and hemicellulose, antibacterial defense strategies (eg, lysozyme), pathogenesis (eg, viral neuraminidase) and normal cellular functions (eg, N-linked glycoprotein biosynthesis). It is a very common enzyme that plays a role in the trimming of mannosidases involved in In bacteria and prokaryotes, glycosidases are known as intracellular and extracellular enzymes and are primarily involved in nutrient acquisition. One of the important glycosidase appearances in bacteria is the enzyme beta-galactosidase (LacZ) involved in the control of lactose operon expression in E. coli. In higher organisms, glycosidases are found in the endoplasmic reticulum and Golgi involved in the processing of N-linked glycoproteins and in lysosomes as enzymes involved in the degradation of sugar chain structures. A deficiency of a specific lysosomal glycosidase causes various lysosomal storage disorders, resulting in developmental disorders or even death. Glycosidases are involved in glycogen biosynthesis and degradation in the body. Together with glycosyltransferases, glycosidases form the main catalytic mechanism for the synthesis and cleavage of glycosidic bonds.

  Diaphorase is a ubiquitous flavin-binding enzyme that catalyzes the reduction of various compounds from the reduced forms of di- and tri-phosphate pyridine nucleotides, that is, NADH and NADPH, and serves as a hydrogen acceptor. Cellular energy metabolism is a complex process that enables cellular energy storage through a series of enzymatic and chemical reactions. One important aspect of cellular energy metabolism is the cellular oxygen reduction state. Live cell metabolic status and enzyme activity and / or metabolic level assays can be determined by measuring the redox defined cofactor NAD (P) / NAD (P) H.

In one embodiment, the present invention provides a compound of formula (I)
Provide
Wherein R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ^{2 ′} is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is an C _{1 ~ 4} alkyl, substituted C _{1 ~ 4} alkyl, -CH ₂ -R ^C, or CH ₂ -V-R ^C;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl, substituted heteroaryl;
L is a linker;
n is 0-3;
R are each independently a C 1 _{~ 7} alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is —S— or —O—; and the dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated.

In another aspect, the present invention provides a compound of formula (II)
Provide
In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹¹ is selected from the group consisting of peptides, amino acids, sugars, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L ′ is a direct bond or a linker;
n is 0-3;
R are each independently a C 1 _{~ 7} alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is —S— or —O—; and the dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated.

In a further aspect, the present invention provides a compound of formula (III)
Provide
Wherein R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹² is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L is a direct bond or a linker;
V is -S- or -O-; and X is each independently -S-, -O- or -NH-;
R are each independently a C 1 _{~ 7} alkyl; and dashed bond indicates the presence of the selectable ring, the ring may be saturated, or unsaturated.

In another aspect, the present invention provides a compound of formula (IV)
Provide
Wherein R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹⁶ is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl, or substituted heteroaryl;
L ′ is a direct bond or a linker;
n is 0-3;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-;
Each X is independently -S-, -O-, or -NH-;
R are each independently a C 1 _{~ 7} alkyl; and dashed bond indicates the presence of the selectable ring, the ring may be saturated, or unsaturated.

  The present invention also provides a method of using the above compound.

It is a figure which shows the result of the biological test of z-DEVD-coelenterazine-h. It is a figure which shows the result of the caspase 3 assay using the oproforus derivative luciferase and z-DEVD-coelenterazine-h. FIG. 2 shows a suitable linker. FIG. 3 shows coelenterazine that can be derivatized. FIG. 5 shows examples of pro-coelenterazine saccharides useful for glycosidase assays. FIG. 2 shows enzymes of various peptide substrates. FIG. 5 shows pro-coelenterazine suitable for use in protease assays. It is a figure which shows the linear correlation of the number of cells and light emission showing that there exists a direct relationship between the light emission measured by the compounds 40 and 50, and the cell number. FIG. 4 is a diagram showing detection of NADH using pro-coelenterazine according to the present invention. FIG. 4 shows another suitable pro-coelenterazine substrate according to the present invention.

  The present invention provides compounds and methods for assaying the presence and activity of various enzymes in a sample.

  Unless expressly specified otherwise, the term “comprising”, as used herein, is intended to mean other things than those listed by “comprising”. It can be optionally present. However, in certain embodiments of the invention, the term “comprising” encompasses the possibility that there is no other, ie, for the purposes of this embodiment, “comprising” Is intended to be understood as having the meaning of “consisting of”.

  As used herein, the following terms and expressions have the meanings specified. It will be understood that the compounds of the invention contain asymmetrically substituted carbon atoms and can be isolated as optically active or racemic forms. Methods for producing optically active forms are well known in the art and are produced, for example, by racemic decomposition or by synthesis from optically active starting materials. All chiral isomers, diastereoisomers, racemates and all geometric isomers of structures are included in the present invention.

  Specific values for radicals, substituents and ranges described below are for illustrative purposes only. It does not exclude other defined values or other values within defined ranges of radicals and substituents.

(Definition)
The term “alkyl” refers to a monovalent moiety obtained by removing a hydrogen atom from a hydrocarbon compound. Alkyl groups include 1 to 30 carbon atoms, or 1 to 12 carbon atoms, or 1 to 10 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms May be. Alkyl groups can be straight or branched and can be saturated, partially unsaturated, or fully unsaturated. The alkyl group may optionally be substituted, for example with halo. Examples of straight chain alkyl groups include, but are not limited to, ethyl, n-propyl, n-butyl, and n-propyl, n-hexyl and n-heptyl. Examples of straight chain unsaturated alkyl groups having one or more carbon-carbon double bonds include ethenyl (vinyl, —CH═CH ₂ ), 2-propenyl (allyl, —CH—CH═CH ₂ ). , And butenyl. Examples of unsaturated alkyls containing one or more carbon-carbon triple bonds include, but are not limited to, ethynyl and 2-propynyl (propargyl). Examples of branched alkyl groups include isopropyl, iso-butyl, sec-butyl, t-butyl and iso-pentyl.

  The term “amino acid” refers to both natural and unnatural amino acids. Also included are protected natural amino acids and protected unnatural amino acids.

The term “aryl” refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring. The aryl group may have _{6 to 10} carbon atoms (C 6-10 aryl). For example, the aryl group may be phenyl or naphthyl.

  The term “halo” refers to a halogen such as Cl, F, Br or I.

The term “heteroaryl” refers to a monovalent moiety obtained by removing a hydrogen atom from a heteroaromatic ring. Heteroaromatic ring, the use of 5 to 10 ring atoms (C 5 _{~ 10} heteroaryl ( "C" atoms are C, N, regardless of which is either O or S, the total number of ring atoms It may be understood to mean))). The ring atom may be carbon, nitrogen, sulfur or oxygen. Multiple heteroatoms may be present in the ring. For example, the heteroaryl group may be furyl, thienyl, thiazolyl, pyrazolyl, triazolyl or tetrazolyl.

  The term “linker” refers to a chain of 2-50 atoms that links a substrate moiety to a coelenterazine nucleus. The linker may include one or more heteroatoms. The linker may also be substituted with an oxo group, amino group, alkyl group, halogen group and nitro group. The linker can also include an aryl group. Suitable linkers include those shown in FIG. 3, such as a p-aminobenzyl linker. Suitably, the linker is a “traceless” or “self-immolative” linker. A “traceless linker” or “self-degradable linker” means that the substrate portion is cleaved from the linker, and the linker spontaneously cleaves from the coelenterazine nucleus, releasing coelenterazine. Representative examples of “self-degradable linkers” include those shown in FIG.

  The term “lower cycloalkyl” refers to a monovalent moiety obtained by removing a hydrogen atom from a hydrocarbon compound having from 3 to 6 carbon atoms. Examples of saturated lower cycloalkyl groups include, but are not limited to groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of unsaturated lower cycloalkyl groups having one or more carbon-carbon double bonds include, but are not limited to, groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and the like.

  The term “luminescent enzyme” refers to a naturally occurring, recombinant or mutated enzyme that uses coelenterazine as a substrate, unless otherwise specified. In the case of naturally occurring luminescent enzymes, those skilled in the art can easily obtain them from living organisms. If the luminescent enzyme is naturally occurring or is a recombinant or mutant luminescent enzyme, that is, if it retains activity in the luciferase-coelenterazine reaction of the naturally occurring luminescent enzyme, bacteria, yeast, mammalian cells, It can be easily obtained from cultures of insect cells, plant cells, etc., and can be converted to express a nucleic acid encoding a luminescent enzyme. In addition, recombinant or mutant luminescent enzymes can be derived from in vitro cell release systems using nucleic acids encoding luciferase. Suitable luminescent enzymes include luciferases derived from bioluminescent decapoda, such as luciferases derived from Oplophoridea, such as luciferases from Oplophorus-derived luciferase, eg, Renilla Luciferase), Alistidae, Solenoceridae, Luciferidae, Shrimpidae, Pasipheidae and Thalassocaridae decapod family and other luciferases derived from marine organisms, and photoproteins such as aequorin.

  The “luminescent reaction mixture” contains a substance that can generate a light signal, that is, luminescence, by a luminescent enzyme. The mixture also contains an enzyme. The required concentration and the specific concentration and / or amount of material required to generate a luminescent signal will vary depending on the luminescent enzyme used and the type of assay. In many cases, other substances are added to the solution. For example, other materials include buffers to maintain the reaction at the proper pH, additives such as PRIONEX or bovine serum albumin (BSA) that help maintain enzyme activity, reducing agents, detergents, etc. .

  The term “peptide” refers to a sequence of at least two amino acids. In some embodiments, the peptide may contain no more than 80 amino acids, or no more than 35 amino acids, or no more than 10 amino acids.

The term “saccharide” refers to a sugar or other carbohydrate , especially a simple sugar. Both α- and β-anomers are included. Saccharide, C ₆ - polyhydroxy compounds, typically C ₆ - be a pentahydroxy often may be cyclic glycal. Included are known simple sugars and derivatives thereof, as well as polysaccharides having two or more monosaccharide residues. Sugars can include protecting groups on the hydroxyl groups. The hydroxyl group of the saccharide can be substituted with one or more acetamide, halo, or amino groups. Furthermore, one or more carbon atoms can be oxidized to, for example, a keto group or a carbonyl group. Suitable sugars include galactose, glucose, glucuronic acid and neurominic acid.

The term “substituted” refers to one or more of the groups represented by the expression “substituted” (eg, 1, 2, 3, 4 or 5, in some embodiments 1, 2, Or 3, and in other embodiments, indicate that 1 or h2) hydrogen is substituted with a group selected from the specified group (s) or suitable groups known to those skilled in the art. Intended. However, the normal valence of the specified atom is not exceeded, and the compound becomes stable as a result of substitution. Suitable substituents include halo, hydroxy, _{phenyl, -NH 2, -NHMe, -NMe 2} , -SH, -CH (OMe) 2, -CF 3, -OCH 3, -SCH 3, C 1 ~ 4 Included are piperazinyl substituted with alkyl, piperazinyl, and aryl.

(Compound)
Coelenterazine is known to emit light when a wide variety of bioluminescent proteins such as marine luciferase act on it. Examples of marine luciferases include Renilla luciferase, Aequorin, Gaussia luciferase, Oprofor luciferase, and Cypridina luciferase.

  The present invention provides coelenterazine derivatives that are non-luminescent enzyme substrates and pro-substrates of photoproteins. Once the non-luminescent enzyme of interest acts, the derivative becomes a substrate for the photoprotein and can therefore be detected by methods known to those skilled in the art.

In some embodiments, the derivative is a compound of Formulas I through IV shown below; (1)
In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ^{2 ′} is selected from the group consisting of peptides, amino acids, sugars, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ , or —NHC (O) OR ^A ;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl, or substituted heteroaryl;
L is a linker;
n is 0-3;
R are each independently a C _{1 ~ 7} alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-; and the dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated;
(2)
In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹¹ is selected from the group consisting of peptides, amino acids, sugars, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L ′ is a direct bond or a linker;
n is 0-3;
R are each independently a C 1 _{~ 7} alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-; and the dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated;
(3)
Wherein R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹² is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L ′ is a direct bond or a linker;
V is -S- or -O-;
Each X is independently -S-, -O-, or -NH-;
R are each independently a C 1 _{~ 7} alkyl; and dashed bond indicates the presence of the selectable ring, the ring may be saturated, be unsaturated;
(4)
Wherein R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁸ is
Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹⁶ is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L ′ is a direct bond or a linker;
n is 0-3;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-;
Each X is independently -S-, -O-, or -NH-;
R are each independently a C 1 _{~ 7} alkyl; and dashed bond indicates the presence of the selectable ring, the ring may be saturated, or unsaturated.

In some embodiments, R ² is
Or, it is a C 2 _{~ 5} alkyl;
Each X is independently -S-, -O-, or -NH-;
Z is —CH— or —N—; Y is —H or —OH; W is —NH ₂ , halo, —OH, —NHC (O) R, —CO ₂ R; and R is it is a C 1 _{~ 7} alkyl.

In some embodiments, R ² is
And; and
X is O or S. In another embodiment, R ² is C _{2 ~ 5} linear alkyl. In certain embodiments, R ⁸ is
Low cycloalkyl, or H, wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl. In another embodiment, R ⁸ is benzyl.

  In some embodiments, V is S.

  Suitable compounds of the present invention are naturally occurring (“natural”) coelenterazine and analogs thereof, eg, WO 2003/040100 and US patent applications, which are hereby incorporated by reference in their entirety. In addition to those disclosed in No. 12 / 056,073 (paragraph “0086”), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e Coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, and 2-methylcoelenterazine. Additional suitable coelenterazines that can be derivatized in accordance with the present invention include those shown in FIG.

  In some embodiments, the coelenterazine derivatives of the present invention are glycosidase substrates. Suitable derivatives include those shown in FIG.

  In some embodiments, the coelenterazine derivative of the present invention comprises caspase 2, caspase 3/7, caspase 6, caspase 8, caspase 9, dipeptidyl peptidase 4 (DPPIV), calpain, chymotrypsin-like proteasome, trypsin-like proteasome, caspase Like proteasome, granzyme B, cathepsin B / L / K, thrombin, trypsin, aminopeptidase, and substrates for proteases such as SARS protease. Suitable peptides and amino acids and the enzymes on which they are substrates include those listed in FIG. Suitable additional derivatives include those listed in FIG.

  In some embodiments, the coelenterazine derivatives of the present invention are substrates for cytochrome P450 enzymes.

  In some embodiments, the coelenterazine derivatives of the present invention are substrates for diaphorase enzymes.

(How to Use)
The coelenterazine derivatives of the present invention can be used in assay reagents to detect the presence or activity of non-luminescent enzymes such as cytochrome P450 enzymes, proteases or glycosidases. Assays using luminescent enzymes and their substrates are well known in the art. For example, a luminescent enzyme, a luminescent reaction mixture, and a coelenterazine derivative that is a substrate of a non-luminescent enzyme may be added to a sample that appears to contain a non-luminescent enzyme. If a non-luminescent enzyme is present in the sample, the non-luminescent enzyme acts on the coelenterazine derivative and is recognized by the luminescent enzyme, producing a luminescent signal. Alternatively, a non-luminescent enzyme can convert the luminescent coelenterazine derivative into a non-luminescent form, ie a loss of signal assay.

  In some embodiments, the chemiluminescence of coelenterazine may be utilized in the assay. For example, a coelenterazine derivative that is a substrate for a non-luminescent enzyme may be added to a sample that appears to contain a non-luminescent enzyme. If a non-luminescent enzyme is present in the sample, the non-luminescent enzyme acts on the coelenterazine derivative, resulting in chemiluminescence, which can be detected by known techniques.

  The coelenterazine derivative may be added to the sample before or simultaneously with the luminescent enzyme. In certain embodiments, the sample may be a cell. The cells may be eukaryotic cells, for example, mammals including but not limited to yeast, avian cells, plant cells, insect cells, monkeys, mice, dogs, cows, horses, cats, sheep, goats or pig cells. It can be a cell, or a prokaryotic cell, or a cell from two or more different organisms, or it can be a cell solution or its supernatant. The cells may be genetically modified by recombinant techniques. In some embodiments, the cells may be from animals such as transgenics, or may be physiological fluids such as blood, plasma, urine, mucus secretions.

  The sample may contain a plurality of non-luminescent enzymes that can be detected. In this case, multiple luminescent enzymes can be used. In addition, multiple substrates can be used. Multiple substrates and / or luminescent enzymes may be used to detect multiple non-luminescent enzymes or other molecule (s) of interest, eg test compounds, simultaneously, eg in various reactions.

  Coelenterazine derivatives are also useful in in situ methods for analyzing cells. Methods for performing in situ analysis of cells using luciferase are known in the art, see for example, US Pat. No. 5,998,204. Coelenterazine derivatives are not luminescent enzyme substrates prior to exposure to non-luminescent enzymes. However, upon exposure to a non-luminescent enzyme, the derivative converts to a compound that can be easily detected in a luminescent reaction in the presence of the luminescent enzyme. Thus, in situ imaging can determine where the non-luminescent enzyme is located in the cell. This can be done by contacting cells expressing the luminescent enzyme with a coelenterazine derivative.

  Alternatively, a transgenic animal expressing a luminescent enzyme gene can be administered with a coelenterazine derivative that is a substrate for a particular non-luminescent enzyme of interest. Thereafter, imaging techniques (eg, in vivo biophotonic imaging) can be used to measure luminescence at the site of luminescent enzyme expression in living intact animals. Thus, a transgenic animal expressing a luminescent enzyme can be administered a coelenterazine derivative that converts to a substrate for the luminescent enzyme in a tissue in which the subject non-luminescent enzyme is expressed. Also, if the luminescent enzyme is expressed in tissue, a luminescent signal is produced and can be detected by any suitable means. Thus, test compounds such as drugs can be tested in animal-based assays. The test compound should be administered to the animal prior to the coelenterazine derivative. Alternatively, tissue from transgenic animals can be used in tissue-based assays.

  In some embodiments, non-transgenic animals may be administered a coelenterazine derivative that is a substrate for a particular non-luminescent enzyme of interest. The derivative converts to a substrate for the luminescent enzyme in tissues where the appropriate non-luminescent enzyme is expressed. Biological samples such as blood, serum, bile, urine, stool, or tissue can be obtained from an animal and can be contacted with a luminescent enzyme. The resulting signal can be detected by any suitable means. Thus, test compounds such as drugs can be tested in animal-based assays. The test compound should be administered to the animal prior to the coelenterazine derivative.

  In some embodiments, test compounds such as candidate agents can be screened and their activity assessed. For example, (1) a non-luciferase enzyme substrate, (2) a non-luciferase enzyme inhibitor, inducer or activator or other regulator, or (3) a cellular condition (eg, increased viability, reactive oxygen species, Or a regulator of an increase in reducibility). Coelenterazine derivatives can also be used to distinguish between substrates and non-luciferase enzymes. Screening can be done either in vitro or in vivo.

  Further, for any of the bioluminescent assays described herein, other reagents may be added to the reaction mixture, including but not limited to those that inhibit or prevent inactivation of luciferase. For example, the emission signal may be expanded or enhanced.

(kit)
The present invention also provides kits for determining the presence or activity of one or more non-luciferase enzymes. The kit can include one or more of the following. That is, coelenterazine derivative (s), non-luciferase enzyme (s), coelenterazine-dependent luminescent enzyme (s), and reaction buffer (s). Reaction buffers may be present in individual formulations for non-luciferase enzyme reactions and luminescent enzyme reactions, or may be present in a single formulation of a single step assay. The kits of the invention can also include inhibitors, activators and / or enhancers of non-luciferase enzyme (s). The kits of the invention can also include assay positive and / or negative controls.

  The invention is further described by the following non-limiting examples.

Example 1: Synthesis of z-DEVD-coelenterazine-h (Compound 21)

tert-Butyl 3-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-((4- (hydroxymethyl) phenyl) amino) -4-oxobutanoate (13):
A flask was charged with Fmoc-Asp (OtBu) -OH (6.7 g, 16.28 mmol) and 100 ml of dry THF. To this solution was added anhydrous N-methylmorpholine (1.9 mL, 17.28 mmol) at ambient temperature and the solution was cooled to −20 ° C. Anhydrous isobutyl chloroformate (2.2 mL, 16.96 mmol) was added via syringe and the resulting suspension was stirred for 10 minutes. Amino alcohol (2.0 g, 16.24 mmol) was added in one portion and the reaction mixture was stirred for 10 minutes, then removed from the cold bath and continued to stir at ambient temperature for 2 hours. The reaction mixture was diluted with 300 mL of ethyl acetate and the mixture was washed successively with 2 × 50 mL water and 2 × 50 mL 0.5 M HCl, 50 mL brine solution. The organic phase was dried (MgSO ₄ ) and concentrated under vacuum. The residue was chromatographed on silica using a heptane / ethyl acetate gradient. This gave 5.2 g (10.7 mmol) of product as a white solid. ¹ H NMR (300 MHz, dmso), d10.02 (s, 1H), 7.89 (d, J = 7.5, 2H), 7.81 to 7.67 (m, 3H), 7.55 ( d, J = 8.4, 2H), 7.47-7.27 (m, 4H), 7.24 (d, J = 8.4, 2H), 5.09 (t, J = 5.7) 1H), 4.51 (m, 1H), 4.43 (d, J = 5.7, 2H), 4.37 to 4.16 (m, 3H), 2.76 to 2.51 (m) 2H), 1.37 (s, 9H).

tert-Butyl 3-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-((4- (chloromethyl) phenyl) amino) -4-oxobutanoate (14):
The flask was charged with alcohol (13) (0.5 mg, 0.97 mmol) and 15 mL of dry benzene. To this solution was added anhydrous triethylamine at ambient temperature and after stirring for 2 minutes, the mixture was cooled in an ice / water bath for 10 minutes. To this was added anhydrous thionyl chloride (78 μL, 1.07 mmol) and after 2 minutes the cold bath was removed and stirring was continued for 3 hours at ambient temperature. The reaction mixture was filtered through a celite pad and the filter cake was rinsed with 30 mL of dry benzene. The filtrate was concentrated under vacuum and the residue was purified by silica column chromatography using methylene chloride. This gave 457 mg (0.85 mmol) of product as a sticky white solid. ¹ H NMR (300 MHz, dmso) δ 10.16 (s, 1H), 7.89 (d, J = 7.5, 2H), 7.79 (d, J = 8.1, 1H), 7.76 ˜7.68 (m, 2H), 7.61 (d, J = 8.5, 2H), 7.44 to 7.27 (m, 6H), 4.72 (s, 2H), 4.58 -4.44 (m, 1H), 4.33-4.18 (m, 3H), 2.63 (ddd, J = 6.0, 14.6, 21.8, 2H), 1.37 ( s, 9H).

Compounds 17 and 18:
A dry flask was charged with coelenterazine-h (7) (200 mg, .37 mmol) and 15 mL anhydrous, deoxygenated DMF under an argon atmosphere. To this solution was added potassium iodide (31 mg, 0.19 mmol) and potassium carbonate (51 mg, 0.37 mmol) at ambient temperature and the mixture was stirred for about 2 hours. To this, compound 14 was added and the mixture was stirred for 18 hours. The product was purified by both reverse phase and normal phase chromatography.

Compounds 19 and 20:
To a solution of either compound 17 or 18 (365 mg, 0.4 mmol) in 3 mL of dichloromethane at ambient temperature was added a solution of 7 mL of dichloromethane in piperidine (2 mL). The reaction mixture was stirred for 30 minutes, diluted with 50 mL toluene and concentrated in vacuo. The residue was purified by reverse phase chromatography using a gradient of 0.1% TFA / water and acetonitrile to acetonitrile. A mixture of regioisomers (133 mg, 0.19 mmol) was decomposed with 2 mL dry DMF and HOBt (30 mg, 0.22 mmol) and z-DEVD-CO ₂ H (140 mg, 0.23 mmol) were added. To this solution was added EDC (42 mg, 0.22 mmol) at ambient temperature and the reaction mixture was stirred overnight. The crude reaction mixture was diluted with 1 mL of acetonitrile and purified by reverse phase column chromatography using a gradient of 20 mM ammonium acetate and acetonitrile. This gave 54 mg of compound 19 and 69 mg of compound 20.

Compound 21:
The crude mixture of compounds 19 and 20 is decomposed with 2 mL dichloromethane, and at ambient temperature this solution contains 4 mL trifluoroacetic acid, 2 mL dichloromethane, 0.4 mL triisopropylsilane, and 0.4 mL thioanisole. Deprotected cocktail was added. The reaction mixture was stirred for 3 hours and diluted with 30 mL diethyl ether. The suspension was centrifuged and the solid was triturated twice with 10 mL diethyl ether. The residue was digested with 2 mL of methanol and purified by reverse phase column chromatography using a gradient of 20 mM ammonium acetate and acetonitrile. This gave 9 mg of compound 21 as an orange solid.

Example 2: Biological test of z-DEVD-coelenterazine-h (Compound 21) Compound 21 (50 μM) was treated with Renilla in 50 mM HEPES buffer pH 7.2, 2 mM DTT, and 0.5% CHAPS. It was mixed with luciferase (6.5 μg / ml, Renilla-GST fusion) and incubated for 20 minutes to remove any free coelenterazine derivative that could be a substrate for Renilla luciferase. Purified caspase-3 enzyme (50 U / ml) or buffer control was used with a final concentration of 25 μM each of Z-DEVD-coelenterazine h derivative substrate, 25 U / ml caspase-3 and 3.25 ug / ml luciferase 1: A volume ratio of 1 was added. Luminescence was measured over 110 minutes. Over time, a 100-fold increase in luminescence was observed as compared to the control sample containing no caspase, which indicates coelenterazine enzyme release, and the coelenterazine compound of the present invention is a caspase enzyme (FIG. 1). It is demonstrated that it can be used for the detection of).

Example 3 Alternative Synthesis of z-DEVD-Coelenterazine-h (Compound 21)

(R) -tert-butyl 3-amino-4- (4- (hydroxymethyl) phenyl) -4-oxobutanoate (C)
B (6 g, 0.011 mole) was decomposed with dimethylformamide (60 mL) in a 250 mL round flask. Piperidine (10 mL) was added. After 4 hours, TLC showed the reaction was complete. The solvent was evaporated. The residue was purified by loading onto celite (15 g in 250 mL EtOAc) and eluting on silica (80 g) with DCM gradient to MeOH (10% in DCM). The combined fractions were evaporated. Yield (76%); Rf = 0.2 (50/50 Hept / EtOAc), 1H NMR (300 MHz, dmso) δ 7.56 (d, J = 8.3, 2H), 7.22 (d, J = 8.3, 2H), 5.06 (t, J = 5.4, 1H), 4.41 (d, J = 4.6, 2H), 3.61 (t, J = 6.5, 1H) ), 2.59 (dd, J = 5.8, 15.5, 1H), 2.41 (dd, J = 7.2, 15.6, 1H), 1.35 (s, 9H); m / Z.

(5S, 8S, 11R, 14R) -tert-butyl 5- (2-tert-butoxy-2-oxoethyl) -8- (3-tert-butoxy-3-oxopropyl) -14- (4- (hydroxymethyl) ) Phenylcarbamoyl) -11-isopropyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazahexadecane-16-oate d)
To a 100 mL round bottom flask, ZD (tBu) E (tBu) V-OH (3.96 g, 6.5 mmol), c (1.6 g, 5.4 mmol), HOBt (0.915 g, 5.9 mmol). ), And DMF (25 mL). To the stirring solution, EDAC (1.15 g, 5.9 mmol) was added. After 18 hours, the solvent was evaporated and the residue was re-decomposed with EtOAc (200 mL). The solution of citric acid (30%, 50mL), washed with bicarbonate (bicarbonate _sat) (50mL), water (2 × 50 mL) and brine (50 mL). The material was adsorbed on celite (15 g) and purified on silica, gradient from heptane to EtOAc. Yield (74%). ¹ H NMR (300 MHz, dmso) δ 9.85 (s, 1H), 8.33 (d, J = 7.7, 1H), 7.95 (d, J = 8.1, 1H), 7.83 (D, J = 8.3, 1H), 7.59 (d, J = 8.3, 1H), 7.53 (d, J = 8.5, 2H), 7.43-7.26 ( m, 5H), 7.21 (d, J = 8.6, 2H), 5.06 (t, J = 5.7, 1H (disappears on D2O shake)), 5.02 (d, J = 4.7, 2H), 4.70 (q, J = 7.7, 1H), 4.41 (d, J = 5.6, 2H (collapse to s on D2O shake)), 4.38- 4.25 (m, 2H), 4.17 to 4.09 (m, 1H), 2.79 to 2.63 (m, 1H), 2.62 to 2.35 (m, 4H), 2. 26-2.11 (m, 2H), 2.00-1. 9 (m, 2H), 1.73 (d, J = 6.0, 1H), 1.34 (dd, J = 4.7, 7.4, 31H), 0.81 (t, J = 6) .3, 6H). MS: measurement value of _{C 45 H 65 N 5 O 13} 883.5; Found 883.7.

(5S, 8S, 11R, 14R) -tert-butyl 5- (2-tert-butoxy-2-oxoethyl) -8- (3-tert-butoxy-3-oxopropyl) -14- (4- (chloromethyl ) Phenylcarbamoyl) -11-isopropyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazahexadecane-16-oate (e)
To a 100 mL RB flask was added d (3.56 g, 4 mmol) along with benzene (50 mL). The solution was evaporated and accumulated overnight under high vacuum. Dry benzene (70 mL) was added to the solid along with TEA (610 μL, 4.4 mmol). The solution was ice-cooled in an ice bath and SOCl ₂ (320 μL, 4.4 mmol) was added slowly. The solution was left to reach room temperature. After 4 hours, additional TEA (241 μL) and SOCl ₂ (100 μL) were added. After an additional 2 hours, the reaction mixture was adsorbed on celite (10 g) and eluted on silica gradient from DCM to 5% MeOH in DCM. Yield (33%). ¹ H NMR (300 MHz, dmso) δ 10.00 (s, 1H), 8.35 (d, J = 7.8, 1H), 7.95 (d, J = 7.8, 1H), 7.84 (D, J = 8.3, 1H), 7.67 (d, J = 8.6, 3H), 7.41-7.22 (m, 7H), 5.11-4.91 (m, 2H), 4.77 to 4.59 (m, 3H), 4.43 to 4.21 (m, 2H), 4.19 to 4.04 (m, 1H), 3.30 (s, (water )) 2.79-2.32 (m, 4H + DMSO), 2.26-2.07 (m, 2H), 2.00-1.79 (m, 2H), 1.77-1.60 ( m, 1H), 1.40 to 1.24 (m, 27H), 0.81 (t, J = 6.4, 6H).

(5S, 8S, 11R, 14R) -tert-butyl 5- (2-tert-butoxy-2-oxoethyl) -8- (3-tert-butoxy-3-oxopropyl) -14- (4-((2 , 8-Dibenzyl-6- (4-hydroxyphenyl) imidazo [1,2-a] pyrazin-3-yloxy) methyl) phenylcarbamoyl) -11-isopropyl-3,6,9,12-tetraoxo-1-phenyl 2-Oxa-4,7,10,13-tetraazahexadecane-16-oate (20).
To a glass bottle degassed with argon gas containing coelenterazine-h (50 mg, 122 μmol) and K ₂ CO ₃ (34 mg, 245 μmol), a degassed solution prepared by mixing e (133 mg, 147 μmol) with DMF (500 μL) was added. It was. After 2 hours, the reaction was injected directly into PR-HPLC and eluted with a gradient from 0.1% TFA (aq) to acetonitrile. The main peak was isolated and evaporated. Yield (13%). UV: Peaks are 250, 300, and 420 nm. ¹ H NMR (300 MHz, dmso) δ 9.79 (s, 1H), 8.27 (d, J = 7.5, 1H), 7.92 (d, J = 8.0, 1H), 7.85 (D, J = 10.1, 1H), 7.58 (d, J = 8.3, 1H), 7.53 (d, J = 8.7, 2H), 7.45-7.14 ( m, 15H), 7.09 to 6.80 (m, 6H), 6.72 (d, J = 8.8, 2H), 5.13 to 4.90 (m, 2H), 4.73 to 4.53 (m, 1H), 4.22 (dd, J = 5.0, 17.7, 3H), 4.16 to 4.01 (m, 3H (2H after D ₂ O shake)), 3 .25 to 3.01 (m, J = 10.0, 16.5, 4H), 2.76 to 2.53 (m, J = 15.6, 2H), 2.51 to 2.33 (m 2H + DMSO), 2.24 to 2.04 (m, 2 ) 1.99 to 1.77 (m, 2H) 1.76 to 1.61 (m, 1H), 1.40 to 1.07 (m, 27H), 0.89 to 0.62 (m, 6H). MS: C71H84N8O14 calculated was 1272.6; found was 1272.8.

Example 4: Synthesis of 30
In 20 ml of methylene dichloride, coelenterazine acetyl ester (0.50 g, 1.14 mmol), quinone trimethyl lock C2-diamine carbonyl chloride (0.436 g, 1.14 mmol), DMAP (0.139 g, 1.14 mmol) and A mixture of TEA (0.115 g, 1.14 mmol) was stirred overnight at room temperature. The compound was purified by silica chromatography using heptane / ethyl acetate as eluent to give a yield of 20% (0.184 g). ¹ H NMR (300 MHz, CD ₂ Cl ₂ ) δ 8.47 (s, 1H), 8.04 (d, 2H), 7.55 (d, 2H), 7.0 to 7.5 (m, 9H) 4.57 (s, 2H, CH2), 4.16 (s, 2H, CH2), 3.2 to 3.7 (m, 4H, NCH2), 2.7 to 3.2 (m, 8H, 2NCH3 + CH2), 2.30 (s, 3H, CH3), 1.0-2.2 (m, 15H, CH3). MS (m / e): 796.5 (M +).

Example 5: Synthesis of 40

40 was prepared by utilizing a method similar to 30 preparation methods (Example 4). Purification by silica chromatography using heptane / ethyl acetate as eluent gave a yield of 60% (0.32 g). ¹ H NMR (300 MHz, CD2Cl2) δ 8.47 (s, 1H), 8.03 (m, 2H), 7.57 (d, 2H), 7.1 to 7.5 (m, 7H), 6. 33 (s, 1H), 6.17 (s, 1H), 4.58 (s, 2H, CH2), 4.19 (s, 2H, CH2), 3.3 to 3.7 (m, 4H, NCH2), 2.7-3.2 (m, 8H, 2NCH3 + CH2), 1.0-2.2 (m, 15H, CH3). MS (m / e): 728.5 (M +). HPLC accuracy: 90% at 262 nm

Example 6: Synthesis of 50

50 was prepared by utilizing a method similar to the method for preparing PBI4442 (Example 20). Purification by silica chromatography using heptane / ethyl acetate as eluent gave a yield of 17% (0.15 g). ¹ H NMR (300 MHz, CD 2 Cl 2) δ 8.42 (s, 1 H), 7.99 (m, 2 H), 7.57 (d, 2 H), 7.1 to 7.5 (m, 10 H), 4. 58 (s, 2H, CH2), 4.19 (s, 2H, CH2), 3.3 to 3.7 (m, 4H, NCH2), 2.7 to 3.2 (m, 8H, 2NCH3 + CH2), 1.0-2.2 (m, 15H, CH3). MS (m / e): 738.5 (M +). HPLC accuracy: 95% at 262 nm.

Example 7: Measuring metabolically active cells using quinone derivatives This example demonstrates the use of quinine-containing coelenterazine derivatives to determine the amount of metabolically active cells. Although living cells maintain a metabolically active state, it is inevitable that if the cell is damaged, that state is lost. Upon entering a living cell, quinone coelenterazine is reduced to a coelenterazine derivative that is a substrate for luciferase utilizing coelenterazine, such as oproforluciferase or Renilla luciferase. The conversion of quinone coelenterazine is proportional to the number of metabolically active cells and can therefore be measured quantitatively by monitoring the light produced by the luciferase reaction.

Two-fold serial dilutions of Jurkat cells were prepared in PBS and transferred to wells of a 96-well plate, 50 μl per well. Compounds 40 and 50 were diluted with PBS to make 50 μM and 100 μM stocks, respectively. 10 μl of the prepared compound stock was added to the cells and the cells were placed in a 37 ° C., 5% CO ₂ incubator. After culturing for 30 minutes, the cells were removed from the incubator, cooled at room temperature for 10 minutes, and 50 μl of oproforluciferase detection reagent was added directly to the wells. Samples were mixed and incubated at room temperature for 20 minutes and luminescence was measured. FIG. 8 shows a linear correlation between the number of cells and the luminescence, which indicates that there is a direct relationship between the luminescence and the number of cells.

Example 8: Caspase 3 Assay Using Oproforsulfuriferase and Compound 21 Oproforsulfuricase (OgLuc) variant, 9B8opt, was used in a bioluminescent assay using a pro-coelenterazine substrate containing a DEVD caspase-3 cleavage sequence. . Purified caspase-3 enzyme is mixed with a lysate sample of E. coli expressing variant 9B8opt purified using halolink ™ resin (Promega) according to manufacturer's instructions, and 100 mM MES pH 6.0, Contains 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 2 mM DTT, 0.25% TERGITOL® NP-9 (v / v), 0.025% MAZU®, 100 mM Diluted 10-fold with buffer with or without 23.5 μMz-DEVD-coelenterazine-h in HERPES pH 7.5. Caspase-3 enzyme was incubated with lysate samples for 3 hours at room temperature, and luminescence was detected on a Turner MODULS ™ luminometer at various time points. Samples containing only bacterial eluate and samples containing only caspase-3 were used as controls. Three replicates were used. FIG. 2 and Table 1 show that the target enzyme can be detected using the pro-coelenterazine substrate of the present invention.

Another oprophorsulluciferase variant L27V02 (“L27V”) was used in a bioluminescence assay using a pro-coelenterazine substrate containing a DEVD caspase-3 cleavage sequence. Purified caspase-3 enzyme (1 mg / mL) in 100 mM MES pH 6 (50 μL) was added to 50 μL assay buffer (100 mM MES pH 6, 35 mM thiourea, 0.5% TERGITOL® NP-9 ( v / v) 227 nM L27V02 variant in 1 mM CDTA, 2 mM DTT and 150 mM KCl) and 47 μM PBI-3741 (z-DEVD-coelenterazine-h). The reaction was incubated for 3 hours at room temperature and luminescence was detected as described above. The L27V02 variant assay was compared to the firefly luciferase assay, caspase-3 / 7Glo (caspase-Glo, Promega). Table 2 shows that the compounds of the present invention can be used in a bioluminescence assay to detect the enzyme of interest.

Prophetic Example 9: O- (8-Benzyl-2- (furan-2-ylmethyl) -6-phenylimidazo [1,2-a] pyrazin-3 (7H) -on-yl) galactoside (25)

Typical synthesis of the typical β-sugar pro-coelenterazine
(O- (8-Benzyl-2- (furan-2-ylmethyl) -6-phenylimidazo [1,2-a] pyrazin-3 (7H) -on-yl) 3,4,5,6-tetraacetoxy -Β-galactoside)

  A 100 mL round bottom flask is charged with a suitable acetobromo-α sugar (eg, acetobromoα-D-galactose) (1.1 EQ), a suitable coelenterazine (eg, (8-benzyl-2- (furan-2-ylmethyl)- Add 6-phenylimidazo [1,2-a] pyrazin-3 (7H) -one) (1.0 EQ, 100 mg), and trifluoromethanesulfonic acid (67 mg, 1 EQ) and degas with argon for 30 min. Inject 4 ml of dichloromethane and 70 μL of tetramethylurea and stir for 3 hours at room temperature, add 4 mL of acetonitrile and filter the solids by pouring the supernatant onto a preparative 250 × 41 mm C18 RP-HPLC column. By purifying the sample and eluting with a gradient of water and acetonitrile or other suitable solvent To collect the appropriate fractions to. Fraction was evaporated to give compound.

  Add 1 mL of methanol to the sample and cool to 0 ° C. in an ice bath. Thereafter, a solution of potassium methoxide (5.2 EQ) in methanol or THE can be added. After 30 minutes, exactly 5.2 equivalents of acetic acid are added. Thereafter, 3 mL of acetonitrile can be added and the sample can be filtered. Purify the sample by injecting the supernatant onto a preparative 250 x 41 mm C18 RP-HPLC column and collect the appropriate fraction by eluting with a gradient of water and acetonitrile or other suitable solvent. be able to. The fraction can be evaporated to give compound (25).

Prophetic Example 10: Synthesis of various P450 substrates
To a mixture of coelenterazine and 1.1 equivalent potassium carbonate in DMF, 1 equivalent methyl iodide can be added at room temperature under an argon atmosphere. The progress of the reaction can be monitored by thin layer chromatography. As soon as the reaction is complete, the mixture can be cooled for several minutes in an ice bath for several minutes and a reaction volume and an equivalent volume of water can be added. The resulting mixture can be extracted with a suitable organic solvent (eg, ethyl acetate), and the extract is concentrated to produce crude compound 1. The material may be further purified by silica gel chromatography.

One equivalent of p-methoxybenzyl bromide can be added at room temperature under argon atmosphere to a mixture of coelenterazine and 1.1 equivalents of potassium carbonate and potassium iodide in DMF. The progress of the reaction can be monitored by thin layer chromatography and when the reaction is complete, the mixture can be cooled for several minutes in an ice bath for several minutes, after which a reaction volume and an equivalent volume of water can be added. The resulting mixture can be extracted with a suitable organic solvent (eg, ethyl acetate), and the extract is concentrated to produce crude compound 2. The material may be further purified by silica gel chromatography.

3-Benzyl-5-phenylpyrazin-2-amine (6) (1 eq), (E) -3- (4-((3,7-dimethylocta-2,6-dien-1-yl) in THF The mixture of) oxy) phenyl) -2-oxopropanoic acid (5) (1.5 eq) and camphorsulfonic acid (1.5 eq) was completed in the presence of molecular sieves and concentration of the starting material was complete Heat at reflux until The reaction mixture was diluted 3 times by volume with ethyl acetate and washed with water and brine solution. After drying over sodium sulfate, the solvent was removed under vacuum and the residue containing compound 7 was decomposed with dry DMF. The resulting solution is treated with acetic anhydride (1.5 eq) and pyridine (1.5 eq) at room temperature. The progress of the reaction can be monitored by TLC, and once complete, the reaction mixture is cooled in an ice bath for several minutes before adding a reaction volume and an equivalent volume of water. The resulting mixture is extracted with a suitable organic solvent (eg, ethyl acetate) and the extract is concentrated to produce crude compound 8. The material is decomposed with methanol, cooled to about 0 ° C. and treated with sodium borohydride (5-10 equivalents) in small portions until product formation is complete. While cool, the reaction mixture can be treated with an equivalent amount of acetic acid to the hydride molecules previously added, and the reaction mixture can be concentrated in vacuo. Crude compound 3 can be obtained by grinding the residue several times with water. This material can also be purified by silica gel chromatography.

3-Benzyl-5- (4- (3,3-dimethoxypropoxy) phenyl) pyrazin-2-amine (10) (1 equivalent), 2-oxo-3-phenylpropionic acid (9) (1. 5 equivalents) and camphorsulfonic acid (CSA) (1.5 equivalents) are heated at reflux in the presence of molecular sieves until the concentration of the starting material is complete. The reaction mixture was diluted 3 times by volume with ethyl acetate and washed with water and brine solution. After drying over sodium sulfate, the solvent was removed under vacuum and the residue containing 11 was decomposed with dry DMF. The resulting solution is treated with acetic anhydride (1.5 eq) and pyridine (1.5 eq) at room temperature. The progress of the reaction can be monitored by TLC, and once complete, the reaction mixture is cooled in an ice bath for several minutes before adding a reaction volume and an equivalent volume of water. The resulting mixture can be extracted with a suitable organic solvent (eg, ethyl acetate), and the extract is concentrated to produce crude compound 12. The material is decomposed with methanol, cooled to about 0 ° C. and treated with sodium borohydride (5-10 equivalents) in small portions until product formation is complete. While still cooled, the reaction mixture is treated with acetic acid equivalent to the previously added hydride molecule and the reaction mixture is concentrated in vacuo. The residue is triturated several times with water to give crude compound 4. This material can also be purified by silica gel chromatography.

Example 11: Measurement of NADH Using the pro-coelenterazine substrate for diaphorase detection of the present invention, the amount of NADH present in a sample was measured. NADH, diaphorase, pro-coelenterazine substrate, and esterase were combined to detect NADH present in the sample. Pro-coelenterazine utilized by diaphorase was converted to coelenterazine. The amount of coelenterazine produced is detected by the oproforsulfurluciferase variant, and the luminescence produced is proportional to the amount of NADH present in the sample.

For the assay, 10 μL NADH (60 μM to 0 μM ground), 10 μL 12 U / mL diaphorase enzyme in PBS, 10 μL 165 U esterase in PBS, and 10 μL 60 μMPBI-4600 in PBS for 1 hour at room temperature In culture. After culturing, an oproforsulfurluciferase detection reagent (10 mM dilution of MESpH6, 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 1 mM DTT, 0.5%) containing a 10 ⁹ dilution of a variant of oproformulluciferase (L27V) Tergitol and 0.05% Mazu) were added. Luminescence was measured after 5 minutes and 1 hour (FIG. 9). According to the data, NADH in a sample can be measured by detecting an enzyme utilizing NADH using the compound of the present invention.

All publications, patents and patent applications are hereby incorporated by reference. In the foregoing specification, the invention has been described with reference to certain embodiments thereof, but many of the details are for illustration purposes only, and those skilled in the art will recognize that the invention may be added to additional embodiments. It will be appreciated that certain details described herein are feasible and may vary considerably without departing from the basic principles of the invention.
Preferred embodiments of the present invention are as follows.
1. Compound represented by formula (II).

In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is

Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹¹ is selected from the group consisting of peptides, amino acids, sugars, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is, C _{1 to 4} alkyl, substituted C _{1 ~ 4} alkyl, -CH ₂ -R ^C or -CH ₂ -V-R ^C;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl; L ′ is a direct bond or linker;
n is 0-3;
Each R is independently C _1-7 alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-; and
Dashed bonds indicate the presence of a selectable ring, which may be saturated or unsaturated;
Compound.
2. Formula (I)


In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ^{2 ′} is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is


Selected from the group consisting of hydrogen or lower cycloalkyl;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl; L is a linker;
n is 0-3;
Each R is independently C _1-7 alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-; and
The dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated.
3. Compound represented by formula (III).


Wherein R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is


Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹² is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or —NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl; L ′ is a direct bond or linker;
V is -S- or -O-; and
Each X is independently -S-, -O-, or -NH-;
Each R is independently C _1-7 alkyl; and
The dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated.
4). A compound represented by formula (IV).

In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁸ is


Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹⁶ is selected from the group consisting of peptides, amino acids, saccharides, —O—R ^A , —OC (O) O—R ^A , —N (R ^B ) ₂ or NHC (O) OR ^A ;
Wherein R ³ and R ⁴ are both hydrogen or C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl, or substituted heteroaryl;
L ′ is a direct bond or a linker;
n is 0-3;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is -S- or -O-;
Each X is independently -S-, -O-, or -NH-;
Each R is independently C _1-7 alkyl; and
The dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated.
5. Where R ² is


Or C _2-5 linear alkyl;
Each X is independently -S-, -O-, or -NH-;
Z is —CH— or —N—;
Y is -H or -OH;
W is, -NH _2, halo, -OH, be _{-NHC (O) R, -CO 2} R; and
R is _C1-7 alkyl,
5. The compound according to any one of 1, 2 and 4 above.
6). Where R ² is


And; and
X is O or S,
6. The compound according to 5 above.
7). _6. The compound according to 5 above , wherein R ² is C _2-5 linear alkyl.
8). Where R ⁸ is


Low cycloalkyl, or H,
The compound according to any one of 1 to 7 above , wherein R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl.
9. In the formula, R ⁸ is benzyl, or a compound according to any one of the above 1 to 7.
10. 10. The compound according to any one of 1 to 9 above, wherein V is S.
11. Contacting a sample suspected of containing an enzyme with the compound of any one of 1 to 10 above, and
Detecting the luminescence of the sample,
A method for detecting the presence or amount of an enzyme comprising the steps of:
12 The method of claim 11, wherein the luminescence is quantified.
13. The sample is suspected of containing a second enzyme,
Contacting the sample with a second compound comprising a substrate for a second enzyme according to any one of 1 to 10 above, and
Detecting the luminescence of the sample;
12. The method according to 11 above, comprising:
14 Administering the compound according to any one of 1 to 10 to a transgenic animal; and
Detecting luminescence,
A method for detecting the presence of an enzyme in vivo.
15. Administering the compound according to any one of the above 1 to 10 to an animal,
Obtaining a sample from an animal; and
Detecting the luminescence of the sample,
A method for detecting the presence of an enzyme.
16.

A compound selected from the group consisting of:

Claims (12)

Compound represented by formula (II).

In which R ² is — (CH ₂ ) _n —T or C _1-5 alkyl;
R ⁶ is selected from the group consisting of —H, —OH, —NH ₂ , —OC (O) R or —OCH ₂ OC (O) R;
R ⁸ is


Selected from the group consisting of hydrogen or lower cycloalkyl;
R ¹¹ is, peptides, amino acids, - OR ^A, is selected from ^{-OC (O) OR A, -N} (R B) 2 , or -NHC (O) group consisting of OR ^A;
Where R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl;
R ^A is C _1-4 alkyl, substituted C _1-4 alkyl, —CH ₂ —R ^C or —CH ₂ —V—R ^C ;
R ^B is, independently, -H or -R ^A;
R ^C is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L ′ is a linker selected from the following groups:

Where R ^x is independently hydrogen, halogen or NO ₂ ;
R ^Y is independently hydrogen or Me, and R ^Z is independently NH, NMe, O, or S;
n is 0-3;
Each R is independently C _1-7 alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl;
V is —S— or —O—; and the dashed bond indicates the presence of a selectable ring, which may be saturated or unsaturated. Where R ² is


Or C _2-5 linear alkyl;
Each X is independently -S-, -O-, or -NH-;
Z is —CH— or —N—;
Y is -H or -OH;
W is, -NH _2, halo, -OH, be _{-NHC (O) R, -CO 2} R; and
R is _C1-7 alkyl,
The compound of claim 1. Where R ² is

And X is O or S.
The compound according to claim 2. 3. A compound according to claim ² , wherein R2 is _C2-5 straight chain alkyl. Where R ⁸ is


Low cycloalkyl, or H,
The compound according to any one of claims 1 to 4, wherein R ³ and R ⁴ are both hydrogen or both C _1-2 alkyl. The compound according to any one of claims 1 to 4, wherein R ⁸ is benzyl.   The compound according to any one of claims 1 to 4, wherein V is S. Contacting a sample suspected of containing an enzyme with the compound according to any one of claims 1 to 7, and detecting the luminescence of the sample;
A method for detecting the presence or amount of an enzyme comprising the steps of:   The method of claim 8, wherein the luminescence is quantified. The sample is suspected of containing a second enzyme,
Contacting the sample with a second compound containing a substrate for a second enzyme according to any one of claims 1 to 7, and detecting the luminescence of the sample;
The method of claim 8 comprising: Administering the compound according to any one of claims 1 to 7 to a transgenic non-human animal, and detecting luminescence;
A method for detecting the presence of an enzyme in vivo. Administering a compound according to any one of claims 1 to 7 to an animal;
Obtaining a sample from an animal and detecting luminescence of the sample;
A method for detecting the presence of an enzyme.